Interactions between the Amino-and Carboxyl-Terminal Regions of G

Apr 25, 1995 - Bradley M. Denker, Paula M. Boutin, Eva J. Neer. Biochemistry , 1995, 34 (16), pp 5544–5553. DOI: 10.1021/bi00016a028. Publication Da...
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Biochemistry 1995,34, 5544-5553

Interactions between the Amino- and Carboxyl-Terminal Regions of G a Subunits: Analysis of Mutated G W G a i 2 Chimeras? Bradley M. Denker,*q$Paula M. Boutin,’ and Eva J. Neers Renal and Cardiology Divisions, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 021 15 Received November 15, 1994: Revised Manuscript Received January 30, I995@

Receptors activate the G a subunits of heterotrimeric G proteins by binding to the C-terminus and reducing their affinity for bound GDP, therefore promoting exchange of GDP for GTP. Although this general mechanism is the same for all G a subunits, different G a subunits vary in nucleotide binding and hydrolysis even though the residues that make up the guanine nucleotide binding site are virtually identical. We have shown previously that truncation of 14 amino acids from the C-terminus of G a , decreased the apparent affinity for GDP and permitted us to see an activated conformation with GTP [Denker, B. M., et al. (1992) J. Biol. Chem. 267,9998-100021. To test whether mutations in the receptor binding region lead to different phenotypes in closely related G a subunits, we made the equivalent deletions in Gai2, synthesized the proteins in vitro in a rabbit reticulocyte lysate and used the pattem of native tryptic proteolysis as an index of conformation. The phenotype of truncated Gai2 was different from that of truncated Ga,: GDP affinity was reduced, but we could not detect an activated conformation with GTP (although GTPyS activated normally). Analysis of shorter deletions showed that loss of three hydrophobic residues (between 11 and 13 residues from the C-terminus) was responsible for the phenotypes. To define the regions of GCL,and Gaiz that were responsible for their different phenotypes, we used a conserved BamHI site (codon 212) to make chimeras. Each chimera truncated at the C-terminus had the phenotype of the donor of the amino-terminal portion. Both truncated chimeras were activated by GTPySlike wild-type proteins, and both had decreased apparent affinity for GDP. Full-length chimeric subunits behaved like wild-type proteins. The crystal structure of G a t and Gail shows that the three hydrophobic amino acids we have identified make contact with residues in the N- and C-terminal portions of the protein. Our studies point to the importance of the contacts in the N-terminal region (start of ,8 strands 1 and 3) that may stabilize the C-terminal a helix, affect nucleotide binding, and determine the characteristic features of different G a subunits. ABSTRACT:

The heterotrimeric G proteins, made up of G a and GBy subunits, transmit signals from plasma membrane receptors to a variety of intracellular effector enzymes and ion channels. Agonist-liganded hormone receptors cause G a subunits to release GDP. The G a subunits then bind GTP, become activated, and remain activated until their intrinsic GTPase activity hydrolyzes GTP to GDP [reviewed in Neer and Clapham (1988), Bourne et al. (1990, 1991), and Simon et al. (1991)l. Recently, the crystal structures of Gat (transducin a) and G a l have been solved (Noel et al., 1993; Coleman et al., 1994). The amino acid sequences of Gat and Gail subunits are similar to those of the other G a subunits, especially those of the GaJGai family (about 68% identity). Ga, is 69% identical to Gai2,while Gail and Gaz are 88% identical. There is only a single amino acid difference in the binding site between Gail and Gat (Leu instead of Val) at position 273 (Coleman et al., 1994). All 14 amino acids that directly contact the bound guanine nucleotide, and the 6 residues that + This work was supported by National Institutes of Health Clinical Investigator Award DK02110-02 (to B.M.D.) and Grant GM 36239 (to E.J.N.). * Author to whom correspondence should be addressed: Renal Division, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Telephone: 617-732-5850. Fax: 617-732-6392. Renal Division. Cardiology Division. Abstract published in Advance ACS Absrracrs, April 1, 1995.

*

@

interact with it indirectly, are identical in Ga,, Gall, and Gai2. Despite the conservation of amino acids comprising the nucleotide binding site, there are differences in both the structure of the binding site and the guanine nucleotide binding characteristics. For example, Arg- 179, which is important for cleavage of the y phosphate (Figure l), is oriented differently in Gat and G a l (Noel et al., 1993; Coleman et al., 1994). Such differences may have important effects on nucleotide binding. In the GdGi family, Ga, binds GTPyS’ faster than Ga2 (ICapp= 0.27 versus 0.063 min-’) and releases GDP faster (k,ff = 0.19 versus 0.072 min-I). The steady-state GTPase activity of Ga, is faster than G a z (turnover number = 0.27 versus 0.095 min-I) (Ferugson et al., 1986; Linder et al., 1990) although the catalytic constant (kcat) is identical (Linder et al., 1990). There are significant differences in nucleotide binding even within the Gai subfamily where the proteins are 85-90% identical. Gain binds GTPyS faster and releases GDP faster than Gail or Ga,, (Carty et al., 1990; Linder et al., 1990). Since the residues that contact the guanine nucleotide are probably the same in these G a subunits, it is likely that these functional differences arise from subtle variations in the positioning of the residues with respect to the nucleotide.



Abbreviations: G proteins, guanine nucleotide binding proteins; GTPyS , guanosine 5’-(y-thio)triphosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

0006-2960/95/0434-5544$09.00/0 0 1995 American Chemical Society

Functional Differences in Closely Related G a Subunits

GTPase Domain

C Helical Domain

N

U P3

FIGURE1: Model of G a subunit. The coordinates for the crystal structure of Ga, (Noel et al., 1993) were used to generate a threedimensional model with the program Molescript (Kraulis, 1991 ). The C-terminal portion (amino acids 208-349 of transducin) is shaded darker, and the GTPase and helical domains are noted. The N- and C-terminus are marked with N and C, respectively. The crystal structure does not include the first 26 amino acids of transducin or the last amino acid. The and p3 sheets are marked by open arrows. The curved solid arrow indicates the chimera site in helix a 2. The site of tryptic cleavage for GDP-liganded G a subunits is a few amino acids amino-terminal to the chimera site. Cleavage at this site generates approximately 25- and 17-kDa fragments. The solid triangle indicates linker 2, which contains Arg179, which is essential for GTP hydrolysis. The bound GTP can be seen as a stick and ball structure just above linker 2. The site of the 14 amino acid C-terminal deletion is noted with white stripes in the C-terminal helix. We previously reported that deletion of 14 amino acids from the C-terminus of Ga, decreases GDP affinity with little or no change in GTPyS affinity (Denker et al., 1992b). Like a receptor-activated G a subunit, the truncated Ga, rapidly exchanges GDP for GTP and can be activated by GTP. Normally, GTP does not activate the G a subunits in solution because the GDP that is rapidly formed from hydrolysis of GTP remains bound to the active site. We now report that an equivalent deletion of 14 amino acids from the C-terminus of Gaiz also reduces apparent GDP affinity but does not lead to a detectable activated state. Analysis of shorter C-terminal truncations suggests that removal of three well-conserved hydrophobic residues 1 1 13 amino acids from the C-terminus causes the observed phenotypes of Ga, and Gaiz. This result gave us the opportunity to segregate the contributions of different parts of Ga, and G Q ~to the final phenotype of the truncated molecule by analysis of Ga, and G Q chimeras. ~ We deleted 14 amino acids from the C-terminus of each chimera and asked whether the phenotype was determined by the “donor” of the N-terminal or the C-terminal portion. A convenient way to make chimeras is to divide Ga, and Ga,z at a highly conserved BamHI site in the cDNA at codon 212. The BamHI site divides the molecule nearly in half (60% of the sequence from the N-terminal and 40% from the C-terminal portion).’ It divides the residues that make up the guanine nucleotide binding site (see Figure 1) and segregates the two The numbering of amino acids used throughout this paper will be for Ga,.Ga, is 354 amino acids; Gail and Ga,, are 355 amino acids. and transducin is 350 amino acids.

Riochernistn, Vol. 34, No. 16. I995 5545 major regions of sequence difference between G a , and Gal, (amino acids 61-175 and 300-317). The region immediately surrounding the RarnHI site is highly conserved with 12 identical amino acids on the N-terminal side and 5 identical amino acids on the C-terminal side (a single conservative difference is then followed by another 1 1 identical amino acids) in G q , and Gqz. We used the native tryptic cleavage patterns of [%Imethionine-labeled G q , and Gail, made in rabbit reticulocyte lysates as a tool to assay the overall conformation of the G a subunit. This approach allows rapid detection of labeled tryptic peptides and has been extensively utilized for G a subunit analysis (Denker et al.. 1992a). Although in vitro translation is not suitable for some kinds of biochemical analysis, the fact that the protein is radiolabeled makes other analysis simpler. For example, tryptic cleavage patterns of in vitro translated proteins are very sensitive indicators of the conformation of G a subunits. There are over 40 potential tryptic cleavage sites in G q , and Gaiz, most of which are inaccessible in the correctly folded molecule. When GTPyS (a nonhydrolyzable analogue of GTP) is bound to the G q , subunit, proteolysis by trypsin only occurs near lysine 21 and yields a stable 37-kDa fragment. In the presence of GDP or of GTP (which is cleaved to GDP, which then persistently occupies the nucleotide binding site), the 37-kDa fra,oment is further cleaved by trypsin to approximately 25- and 17kDa peptides (Fung & Nash, 1983; Hurley et al., 1984; Winslow et al., 1986). Stabilization of the 37-kDa form is an index of the activated (or GTP-liganded) conformation of G q , and Gaz, while production of the 25- and 17-kDa products reflects the inactive, GDP-liganded state. The tryptic sites that are accessible in the GDP-liganded form are near Arg-209 in a region that changes conformation on GDP binding and is within a few amino acids of the junction site of the chimeric molecules (see Figure I). Our analysis of the properties of wild-type G a subunits and of full-length and truncated chimeras suggests that although the truncations are at the C-terminus, the resultant phenotype is largely determined by the N-terminal portion (amino acids I -2 12). Because the crystal structures of G a subunits are available, we know which residues in the N-terminal portion of G a contact the three critical residues in the C-terminus whose removal leads to the phenotypes we observe. Our studies allow us to suggest which of these residues are most important for normal guanine nucleotide exchange.

EXPERIMENTAL PROCEDURES Mutagenesis and Construction qf Chirneras. Deletions in rat Ga, and rat Ga,’ were generated by the polymerase chain reaction. All PCR products were subcloned into the EcoRV site of Bluescript (Stratagene) by blunt-end ligations. The 5’ and 3’ primers for G a z included Hind111 restriction sites. The cDNAs for these subunits were kindly provided by Dr. R. Reed, The Johns Hopkins University, Baltimore, MD. All mutated cDNAs were sequenced by the dideoxy method (Sanger et al., 1977). The complete amino acid sequences were verified for GG- 14 and G ~ zI4- and were identical to wild-type rat clones outside the mutation. Chimeras of Gq, and G a z were made using a conserved RarnHI site in codon 212 of the amino acid sequence. G a z contains a single BarnHI site, but Gq, has an additional site in codon 136. Gaiz and GQZ-I4 were excised from Blue-

Denker et al.

5546 Biochemistry, Vol. 34, No. 16, 1995 script with HindIII and then digested with BamHI, generating N- and C-terminal fragments. To obtain the correct BamHI fragments from Ga, and Ga,-14, they were excised from Bluescript with EcoRI and SaA. The insert of -1.1 kb was gel purified and digested with BamHI (8 units/pL) at 1 unit/ pg of Ga, cDNA or 0.5, 0.25, or 0.125 unit/pg DNA for 1 h at 37 "C. For Ga, and Ga,-14, five different-sized fragments were generated, and the two corresponding to the expected size of the N- and C-terminal portions were isolated from agarose gels. Chimeras were made by ligating the appropriate fragments together with either HindIIIISaA-cut Bluescript or HindIIVEcoRI-cut Bluescript. Chimeras were confirmed by dideoxy nucleotide sequencing from the Nand C-terminii and by BamHI digests. In Vitro Translation. mRNA was transcribed from the cDNA, and the proteins were made by in vitro translation using a rabbit reticulocyte lysate as described previously (Denker et al., 1992b). Some proteins were in vitro translated from the cDNA using a single-step method [TNT in vitro kit (Promega)] which allows transcription and translation to occur in the same reaction tube. Labeled proteins were analyzed by SDS-PAGE (Laemmli, 1970); gels were soaked in Enhance (Du Pont-New England Nuclear), and autoradiograms were exposed for 1- 10 days with Fuji XAR film and two enhancing screens. Tryptic Digestion. Samples of reticulocyte lysate (5- 10 pL) were incubated with 0-20 pmol of L-1-(tosy1amido)2-phenylethyl chloromethyl ketone-treated trypsin (Cooper Biomedical) at 30 "C for 10 min. Samples were preincubated with either GTPyS, GDP, or GTP at the concentrations stated in the figure captions for 10 min at 30 "C. All experiments with GTPyS and GTP were done by adding nucleotide directly to the translation mixture. The reticulocyte lysate contained -50 pM GTP [information from Promega, Inc. (Surgenor, 1974)l. Experiments with no nucleotide added to the reticulocyte lysate gave tryptic pattems nearly identical to those from experiments done with 100pmol of GTP added (about 150pM total). GTP was added to minimize variations in lysate GTP concentrations. For experiments with GDP, the nucleotides and creatine phosphate in the translation mixture were removed by Sephadex G-50 (Pharmacia LKB Biotechnology, Inc.) column chromatography in 50 mM TrisHCL, pH 7.6, 1 mM dithiothreitol, 1 pM GDP, 1 mM EDTA, and 6 mM MgC12 at 4 "C. Proteolytic reactions were stopped by the addition of SDS-PAGE sample buffer and heating to 100 "C for 3 min. Samples were analyzed by SDSPAGE as described above. Sucrose Density Gradient Centrifigation. Sucrose density gradients (5-20%) were made up in 50 mM Tris-HC1, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 0.3% Triton X-100, 1 mM MgC12, and 100 pM GDP. All gradients contained 100 pg of bovine serum albumin ( S Z O , ~= 4.3), ovalbumin ( s , , , ~= 3 3 , and carbonic anhydrase ( ~ 2 0 = , ~ 2.9) as markers. Ga, and Ga,z were in vitro translated as described above, incubated with 100 pM GDP for 10 min at 30 "C, and then digested with 2 pmol of trypsin for 10 min at 30 "C. Trypsin was inactivated with the addition of 0.1 pmol of benzamidine. Trypsinized Ga, and Gaiz were mixed with undigested in vitro translated wild-type proteins and markers and were centrifuged at 52 000 rpm for 16 h at 4 "C as previously described (Denker et al., 1992b). Gradient fractions were analyzed by SDS-PAGE as described above. The positions of the peaks of marker and radiolabeled proteins were

determined by laser densitometry of autoradiograms or stained gels. Cross Linking. The cross-linking reagent BMH [ 1,6-bis(ma1eimido)hexanel was prepared fresh as a 50 mM stock in dimethyl sulfoxide. Dithiothreitol (DTT) was removed from samples translated in vitro by Sephadex G-50 column chromatography. One-milliliter columns were equilibrated with 50 mM Hepes, pH 8, 6 mM MgC12,75 mM sucrose, 1 mM EDTA, and 0.1% Triton X-100. Twenty-microliter samples were cross-linked in 2 mM BMH on ice for 20 min and then quenched by addition of DTT to 20 mM. Bovine brain purified /3y was added to all samples, and controls had DTT added to the mixture prior to addition of BMH. Samples were analyzed by SDS-PAGE and autoradiography as described above.

RESULTS Deletion of 14Amino Acidsfrom the C-Terminus Produces a DifSerent Phenotype in G a , and Gaiz. As we have previously shown (Denker et al., 1992b), deletion of 14 amino acids from the C-terminus of Ga, decreases GDP affinity. We can detect the activated conformation (determined by stabilization of the 36-kDa ~ e p t i d e of ) ~ Ga,-14 with both GTPyS and GTP (Figure 2a,b). The stabilization of the 36-kDa peptide of Ga,-14 with GTP reflects the reduced affinity for GDP and the relatively high GTP concentration in reticulocyte lysate (see Experimental Procedures) (Surgenor, 1974). However, the proteolytic pattems of Gcg-14 and Gai2-14 with GTP differ strikingly. The 36kDa band does not form from Gai2-14 in GTP at any trypsin concentration (Figure 2d) and cannot be detected even with 10 mM GTP (data not shown). With GTP, trypsin apparently degrades Gai2-14 to small peptides. The failure of Gaiz-14 to be stabilized by GTP is not likely to be due to protein misfolding or to inability to bind GTP since the protein is stabilized by GTPyS (Figure 2c). We see somewhat less GTPyS-stabilized fragment arising from GQZ-14 than from GG-14 at higher amounts of trypsin. This difference probably refects the fact that wild-type Gai2 is intrinsically more sensitive than Ga, to tryptic proteolysis (data not shown). Both wild-type Gai2and Gap14 translated in vitro can interact with /3y to form the expected a-/3 cross-linked products that migrate with apparent molecular mass of 122 and 140 kDa as previously described for Gail and for Ga, (Thomas et al., 1993; Yi et al., 1991) (Figure 3). The concentration range over which GTPyS stabilizes the 36kDa peptide is similar for Ga,, G Q ~ G&-14, , and Gaiz-14 (1-100 pM). Although Ga,-14 is partially and Gai2-14 is almost totally degraded by trypsin with GTP, the characteristic 25-kDa band produced from GDP-liganded G a subunits (see Figures 6 and 7 for examples of wild-type cleavage pattems with GDP) is not seen unless the GDP concentration is increased to 10 mM (Denker et al., 1992b). The faint bands seen with Gcb-14-GDP [shown in Denker et al. (1992b)l are also seen with Gai2-14-GDP (data not shown). Therefore, we conclude that both Ga,-14 and GQZ-14have a significantly reduced ability to bind GDP. Both proteins with C-terminal deletions can bind GTPyS as demonstrated Since about 1 kDa has been removed from the C-terminus, the stable GTPyS and GTP-liganded product is now 36 kDa instead of 37 kDa.

Functional Differences in Closely Related G a Subunits

Riocltemistn, Vol. 34, No. 16, 1995 5547

b

8

kDa

kDa

3836-

Trypsin GTPys

38-1) 36

a-

T r y p s i n 0 .7 + GTP

.7 2 7 2 0

0

-

-

+ + + +

kDa

kDs

3836-

384 36-1-e

-

720

+ +

2 7 20

+ + +

d

C

Trypsin 0 2 GTPyS +

=--- a-

+

T r y p s i n 0 .7 2 7 20 - + + + + GTP

FIGURE 2: Pattems of tryptic digestion of G%-14 and Gaiz-14translated in vitro. In vitro translated Ga+-14 (a, b) and Gqz-14 (c, d) were digested for IO min at 30 "C with increasing amounts of trypsin (0-20 pmol) after incubation with GTPyS (100 pM) (a, c) or GTP (100 p M ) (b, d) as described in Experimental Procedures. The arrows mark the location of stabilized 36-kDa peptide. The smaller band underneath J the starting material of G a p 1 4 (c, d) is most likely an intemal initiation site. Gqz has methionine at amino acid 18 (Ala in G ~ which would give rise to a labeled protein of this molecular weight. We identify this band in all G q z cDNAs, including those modified at the C-terminus: Gqz (Figure 6b), Gqz/Ga+ (Figure 6d), Gai2/G%-14 (Figure Sb), and Gqz-10 and -13 (Figure 4c.d). These observations make proteolytic digestion or premature termination unlikely explanations for the origin of this band.

by protection of the 36-kDa peptide (Figure 2a,c), but only one of them ( G G - I ~ )shows an activated state with GTP. Tlwee Hydrophobic Amino Acids in the C-Terminal Portion Are Important for the Pltenohpe qf Truncated Goc, and Gai2. To narrow the C-terminal amino acids essential for these phenotypes, we deleted 10 and 13 amino acids from the C-terminus. Table I shows the C-terminal sequences of GG and G Q ~ and the sites of the C-terminal deletions. Tryptic proteolytic patterns with GTP of G%- 10 and G ~ z - 1 0are similar to those of full-length subunits (Figure 4a,c compared with Figure 6a,b). However, both G a - I 3 and Gaiz-13 have tryptic patterns with GTP that are indistinguishable from those of proteins missing the last 14 amino acids (Figure 4b,d compared with Figure 2b,d). The tryptic patterns of the 13 and 14 amino acid C-terminal

deletions with GTPyS and GDP are also indistinguishable (data not shown). Both G a - I 3 and Ga2-13 are stabilized with 10-100 pM GTPyS and attain the GDP-liganded conformation poorly only in 10 mM GDP. Therefore, we conclude that the phenotype of both G ~ Gand , G Q is ~ caused by removal of residues 1 1 -13 from the C-terminus (see Table 1 ). These three hydrophobic residues ( 1 1 - I3 from the C-terminus) are highly conserved in all G a subunits. The N-Terminal Portion (AminoAcids 1-212) Determines the Pltenotye qf C-Terminal Truncated Ga Chimeras. To determine whether the phenotypes of G a - 1 4 and GQ~-14 were due to differences in the N- or C-terminal portions of the molecules, we made C-terminal deletions in chimeric G a and G Q subunits. ~ The tryptic pattern observed with proteins missing 14 amino acids from the C-terminus could arise from

5548 Biochemisty, Val. 34, No. 16, 1995

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Table 1: Amino Acid Sequences of C-Terminus of Ga, and Ga,z and Carboxyl-Terminal Deletions"

Gain

. . . . .

A V T D V I

Gain-14

. . . . .

A V T -

G a p 13

. . . . .

A V T D -

Gain- 10

. . . . .

A V T O V I

I

K N N

L K O C G L F

- - - - - -

- - - - - - -

- - - - -

- - - - - - -

- - -

- - - - - - -

I

Dots represent continuation of sequence and dashes represent deleted amino acids. a

b Kda

Kda

=+

140122-

39- rru -

0 -

BMH

-

+

140122-

39-

BMH

!-

-7 -

+

-

+

FIGURE3: Cross-linking of G q z and Gqz-14 to Py subunits. In vitro translated G q z and Gqz-14 were prepared as described in Experimental Procedures. Samples including bovine brain /Jy (0.3 p g ) were incubated with 2 mM BMH (+)or 2 0 mM D I T followed by addition of 2 mM BMH (-) on ice for 20 min as described in Experimental Procedures. Samples were analyzed by SDS-PAGE and autoradiography as previously described. Panel a: cross-linked bovine brain purified G@y stained with Coomassie blue. Panel b: In vitro translated and cross-linked G q z (left) and Gqz-14 (right). The gel was exposed for 24 h at -70 "C with two enhancing screens. The characteristic bands that migrate with apparent molecular masses of 122- and 140-kDa are marked with the arrow. Cross-linking efficiency with in vitro translated proteins is less than that with pure proteins. This is probably due to the high concentration of proteins in rabbit reticulocyte lysate competing for crosslinking reagent.

disruption of amino acid interactions with either the N- or C-terminal portion of the molecule. Like wild-type Get, and G Q ~both , chimeras with C-terminal truncations bind GTPyS and can attain the activated conformation (Figure 5, sets a, b, and c, last lane of each set), showing that the truncated chimeras are functional by this criterion. With GTP, each chimera behaved similarly to the amino-terminal donor; GaJ ~ 2 - 1 4is activated by GTP for a sufficient time to detect a

substantial amount of protein in the GTP-liganded conformation (36-kDa band, Figure 5c). We detect only a very faint 36-kDa band for Ga&,- 14 in the GTP-liganded conformation (Figure 5b). The band is similar in size to a minor band in the starting material that is probably due to an internal start site (see the caption to Figure 2). However, it is stable to tryptic digestion and may represent a very small amount of GTP-liganded G~z/Gct,14. Since both C-terminal truncated chimeras can bind GTPyS and give tryptic cleavage patterns similar to those of wild-type proteins, neither chimera is grossly misfolded (last lane in each set). In addition, like wild-type truncated proteins, both truncated chimeras form faint 25-kDa bands with high (10 mM) GDP (not shown). The truncated chimeras can be cross-linked to By subunits to form the same products as wild-type proteins and nonchimeric truncated proteins (described above and in Figure 3), giving further evidence that they are not grossly misfolded. These results show that the N-terminal donor to the chimera determines the phenotype of proteins with C-terminal deletions. Exchanging the C-Terminal Region Does Not Significantly Affect the Structure of the N-Terminal Region. To ensure that chimera construction per se did not alter the pattern of tryptic proteolysis, cleavage patterns of full-length chimeras GaJGa2 and Gcqt/Gct, were compared with wild-type patterns (Figure 6). Tryptic cleavage patterns of full-length proteins without added nucleotide reflect the GDP-liganded conformation because GTP in the rabbit reticulocyte lysate (see Experimental Procedures) is hydrolyzed to GDP, which remains tightly bound to the active site. Trypsin cleaves near Arg-209, generating 25- and 17-kDa peptides (see Figure 1). The tryptic cleavage pattern of GG-GDP (Figure 6a) differs from that of Ga2-GDP (Figure 6b). Ga-GDP gives a doublet at 25 kDa, and the lower band of the doublet increases with increasing trypsin concentrations (Figure 6a). For Q~-GDP,increasing the trypsin concentration causes cleavage of the 25-kDa band to a doublet at 20 kDa (Figure 6b). A faint 17-kDa band is visible for both Gct, and G Q ~ at the lower trypsin amounts (see Figure 6a,b). The 17-kDa

Functional Differences in Closely Related G a Subunits

Biochemistry, Vol. 34, No. 16, I995 5549

a

b kDa

kDa

38-

36-

--

3836----I-

a-

25-

Trypsin GTP

--a-

0 .7

2 7 20

- +

+ + +

Trypsin GTP

c

0 -7

2

7 20

- + + + +

d kDr

384 364

aa-

2520-

Trypsin GTP

0 .7

2

7 20

- +

+ + +

Trwsin 0 .7 2 7 2 0 GTP - + + + + FIGURE 4: Comparison of tryptic patterns for G ~ cand , Gqz with IO and 13 amino acids deleted from the C-terminus. In vitro translated Gcb-10 (a), GG-13 (b), Gq2-10 (c), and Gqz-13 (d) were incubated with 100 pM GTP followed by tryptic proteolysis as previously described. G c ~ , - 1 3forms the protected 36-kDa band [arrow in (b)] similar to GG-14. The arrow in (d) marks the location expected for stabilized 36-kDa peptide, which is not seen for Gqz- 13 (similar to Gqz- 14).

band is difficult to visualize because it has only three methionines (versus six in the 25-kDa peptide of G h and five in G Q ~ )and is more sensitive to further tryptic proteolysis than the 25-kDa band [even with purified protein from bovine brain (Winslow et al., 1986)J. Despite the strongly interactive N- and C-terminal regions, exchanging C-terminal regions (amino acids 212-354) does not significantly affect protein folding of the amino terminus (25-kDa bands) as detected by tryptic proteolysis. The GaJ GQ tryptic pattern is indistinguishable from that of GG (Figure 6a,c), and G Q ~ / G was ~ nearly identical to wild6 b,d). The characteristic tryptic patterns type G Q (Figure ~ of the wild-type proteins are seen in the chimeric molecules containing the wild-type donor of its amino terminus (Figure 6c,d). This is to be expected since the 25-kDa fragments come from the amino terminus, but it is reassuring that

addition of a heterologous C-terminus does not cause gross changes in the folding of the N-terminal region. G Protein a Subunit Tnptic Fragments Remain Associated aBer Tryptic Digestion. The stabilization of fragments by GDP suggests that the guanine nucleotide binding site of Ga subunits is still intact after tryptic proteolysis. There are 20 amino acids that interact with either the phosphates or the guanine ring of bound nucleotide; 14 are located in the 25-kDa fragment, while six are in the 17-kDa fragment. Sucrose density gradient analysis shows that the 25- and 17kDa tryptic peptides remain associated because they cosediment with undigested, in vitro translated wild-type protein (Figure 7). The ~ 2 0 values . ~ are the same as we previously reported for wild-type in vitro translated GG (3.1 + 0.2, n = 4) for the 39-, 2 5 , and 17-kDa peptides) (Denker et al., 1992b). The pattern for G Q ~ is similar to that of Gee, with

5550 Riochemistn?, Vol. 34, No. 16, 1995

Denker et al.

a

b

C

kDa

Trypsin GTP GTPyS

0 .7 2 7 2 0 2 0 0 .7 2 7 2 0 2 0 0 .7 2 7 2 0 2 0

-. .+- +- -+ .+ . -

+

- - -

- + + + + -

0

-

0

+

0

+ + + + 0

.

0

0

-

+

FIGURE 5: Comparison of tryptic digestion of chimeric G a subunits with C-terminal truncations. In vitro translated Ga,,-I4 (a), G@&,-14 O pM GTP as previously described. (b). and GocJ@z-14 (c) were digested with increasing amounts of trypsin after preincubation with I The last lane of each group shows tryptic digestion with 20 pmol of trypsin after preincubation with I Op M GTPyS. The arrow marks the position of the protected 36-kDa peptide which is seen for G&-I4 (a) and GcCJqz-14 (c) but not for G@~/a,,-l4(b). The faint band seen in this position arises from the internal start site that was described in the Experimental Procedures. Note that G @ & , - l 4 is stabilized with GTPyS (last column in panel b).

+

an ~ 2 0 value . ~ of 3.3 0.2 (n = 6) for the wild-type protein and the 25- and 17-kDa peptides. These results are consistent with our results and the crystal structure of Ga, and G a l , which shows that tryptic sites do not define distinct domains of the G a subunit and that the N- and C-terminal regions of the molecule are strongly interactive through extensive hydrogen bonding (see Figure 1) (Noel et al., 1993).

DISCUSSION Our findings that identical mutations in Gcc, and G Q give ~ different phenotypes allow us to begin to study regions of difference among G a subunits that are likely to explain some of their distinct functions. Figure I shows the threedimensional structure of Ga, with the portion C-terminal to the junction site of the chimeras shaded. The tryptic site accessible in the GDP-liganded conformation occurs several amino acids upstream from the junction site of the chimera (Figure 1). The crystal structure of Ga, and G a l reveals a network of hydrogen bonds that link one functional region to another. Our observation that the 25- and 17-kDa tryptic fragments sediment as a complex on sucrose density gradients is consistent with the integrated structures of transducin and G a l and, by inference, of Gcc, and G Q ~ In . principle, the phenotypes of Gee,- 14 and Gaz- 14 could have been determined by the N-terminal region, the C-terminal region, or both. Our analysis of chimeric truncated molecules did not reveal an intermediate phenotype but showed that the phenotype of the chimera matched that of the aminoterminal donor. We have previously shown that truncation of 14 residues from the C-terminus of Gcc, decreases its apparent affinity for GDP and allows the GTP-liganded state to exist long enough to be detected by the tryptic proteolysis assay, probably because of increased GTP/GDP exchange (Denker et al., 1992b). We now show that the same truncation of Gq, decreases the apparent affinity for GDP (because the characteristic tryptic fragments only appear at very high GDP

concentration),but the GTP-liganded form cannot be detected even at 10 mM GTP. Both truncated forms of Gcc, and Gq, bind GTPyS similarly since they can be stabilized over a similar range of GTPyS concentration ( 1 - 100 pM). AIthough most of our studies were done with G a subunits and chimeras from which 14 amino acids were removed, we have shown that removal of 13 amino acids is sufficient to elicit the full phenotype in both Gq, and G a l . Removal of 10 amino acids from either wild-type GQ-,or Gaiz had only a small effect on the apparent GDP affinity and gave a tryptic proteolytic pattern similar to wild type (Denker et al., 1992b). The three amino acids that are 1 1, 12, and 13 residues from the C-terminus (Ile-Ile-Ile in Gcc, and Val-Ile-Ile in Ga2; see Table 1) are conserved in virtually all G a subunits. The crystal structures of transducin and G a l suggest how loss of these residues may affect the GDP affinity. In transducin, these three hydrophobic residues interact with Val-30, Leu- 190, Cys-2 16, and Tyr-3 16. They are equivalent to Val-34, Leu- 195, Ala-22 I , and Tyr-32 1 in Gcc,, Ga, I , and Gq2. Two of these contact residues fall in the N-terminal, and two in the C-terminal, portion of our chimeras. Since each chimera behaves like the N-terminal donor, our study suggests that disruption of the N-terminal contacts is a major contributor to the final phenotype. The N-terminal contacts are more important than the C-terminal contacts. The two in the amino-terminal portion occur at the start of the /?I and p3 strands (see Figure 1). It is likely that the interaction of the C-terminal helix with these two residues constrains the flexibility of the helix. Eliminating this interaction may make the helix more flexible, causing a conformational change that is propagated to the loop at the end of the a helix. This loop is important for stabilizing the binding of the guanine ring. Such a mechanism is responsible for the activated phenotype of G%-A366S, a mutation that leads to precocious puberty in humans (Iiri et al., 1994). We have previously shown that mutation of a cysteine in this loop to alanine (C325AGcc,) decreases GDP affinity of GQ-,by about an order of magnitude (Thomas et al., 1993). Val-34 that

Functional Differences in Closely Related G a Subunits

Biochemistry Vol. 34, No. 16, 1995 5551

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Trvpsin GTP

0 .7 2 7 2 0

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FIGURE 6: GTP-liganded patterns of tryptic digestion for wild-type Ga,,, Gap, GaJGap, and G@?/Ga,. Ga, (a), G q z (b), GCGJGai2 (c), and Gq?/Ga,, were digested with increasing amounts of trypsin after incubation with GTP ( 100 p M ) as previously described. Panels a and b show the characteristic 20-25-kDa peptides generated from GDP-liganded Gcl, and G q z subunits. The chimeric subunits (c, d) have the same pattern as the donor of the amino terminus. A band at 45 kDa is occasionally seen even in controls without added mRNA (panel b). Its intensity vanes with the manufacturer of the [%]methionine used to label the translated protein, and it is usually absent when we use [S3']methionine from Amersham (Arlington Heights, IL). starts /3 strand 1 (Figure 1) has been mutated to alanine (Slepak et al., 1993). Goc, with this point mutation bound GTPyS and interacted normally with b y , but there is no information about GDP binding. We would predict that mutating both Val-34 and Leu-I95 to bulky polar amino acids would lead to a decrease in GDP affinity. If the contacts with bound nucleotide are really the same in Goc, and G Q as ~ in transducin and G Q ~ then , all the residues that are predicted to contact the deleted hydrophobic residues are identical in Goc, and G Q ~ . The different phenotypes therefore are likely to result from subtle differences in folding within the amino terminus between Goc, and G Q ~that position identical amino acids somewhat differently.

We had originally explained the activation of GG- 14 with GTP by rapid exchange of GDP for GTP so that the GTPliganded state existed for a larger fraction of the time than the wild-type Goc,. Like Ga+-14, G ~ 2 - 1 4also binds GDP poorly, but it does not show an activated state with GTP. Therefore, the difference in phenotype between Got,- I4 and G Q ~ - I 4may reflect an additional differential effect of the truncation on the GTPase activity. The catalytic constant (kcat)is the same for both wild-type GG and G Q (Linder ~ et al., 1990). The C-terminal truncation could decrease the intrinsic GTPase activity of Goc,, keeping GTP in the active site long enough to be detected by the trypsin assay, or increase the GTPase activity of G Q ~14, - shortening the time

5552 Biochemistry, Vol. 34, No. 16, 1995

Denker et al. kDa

kDi

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39

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25'

17-

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I

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I

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BSAOA CA

:;i[

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t t t

A

58

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~~

.c cn

I

I

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a 0

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FIGURE 7: Sucrose gradient sedimentation profile of G a , translated in vitro and trypsinized. In vitro translated G a , (a, left two panels) and Gaz (b, right two panels) were digested with 2 pmol of trypsin after preincubation with IOOpM GDP as described in Experimental Procedures. The reaction was stopped by the addition of 0.1 pmol of benzamidine, and the sample was mixed with uncleaved Ga, or Ga2 translated in vitro and 3 p g of Ga, subunit (purified from bovine brain). The mixture of trypsinized and untrypsinized subunits was centrifuged through 5-2096 sucrose gradients containing 1 O p M GDP. Marker proteins (1OOpg) were included in the sample: bovine serum albumin (BSA), carbonic anhydrase (CA), and ovalbumin (OA). Upper panels: Autoradiograms of fractions from a sucrose density gradient analysis of in vitro translated Ga, (a) or Gai2(b). Lower panels: Densitometry of the autoradiograms shown above. (m) 39-, ( x ) 25-, and (A) 17-kDa peaks. Sedimentation positions of marker proteins are shown by arrows.

GTP is bound and preventing detection by this assay. Ultimately, purified proteins will be needed to evaluate these different potential mechanisms. It is possible to use immunoprecipitation of in vitro translated proteins to measure the amount of bound GDP. However, most precipitating antibodies are against epitopes in the C-terminus and would not be useful for these studies. Furthermore, these measurements ~ the reticulocyte would be confounded by endogenous G Q in lysate. In summary, our studies have shown that the N- and C-terminal portions of GG and G Q have ~ sufficient interactions to remain associated when cleaved by trypsin. The network of hydrogen bonds that stabilize the structure allows conformational changes to be transmitted throughout the molecule. A functional consequence of such an integrated structure is that the phenotype of deletions in the C-terminal portion is determined by the structure of the N-terminal portion. Agonist-liganded hormone receptors interact with the C-terminus of G a subunits to reduce GDP affinity (Vi et al., 1984; Van Dop et al., 1984; West et al., 1985; Hamm et al., 1987, 1988; Sullivan et al., 1987; Masters et al., 1988; Spiegel et al., 1990; Okamoto et al., 1994). Hormone

receptors are thought to affect the GTPase activity of G a subunits by decreasing GDP affinity and not by changing kea,, although the evidence is indirect (Landis et al., 1989). It is possible that the motion of the C-terminal a helix induced by receptors may also cause subtle changes in the GTPase activity of G a subunits. Such differences would lead to variations in the lifetime of the activated state. Our studies suggest that three hydrophobic amino acids near the C-terminus are especially important for inducing the conformational change that allows GDP affinity to fall and G a activation to occur. We predict that mutations of these amino acids would lead to a phenotype similar to those of the C-terminal truncations. The subtle structural differences between GG and G Q ~have significant functional consequences. Therefore, although all the G a subunits are extremely similar, each has its individual character that is likely to suit it for a specific intracellular role.

ACKNOWLEDGMENT The authors wish to thank Drs. Raman Nambudripad and Temple F. Smith for their help with the Molescript program and the generation of Figure 1.

Biochemistry, Vol. 34, No. 16, 1995 5553

Functional Differences in Closely Related G a Subunits

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